Co-culture Of Stem Cells Research Articles

EFFECTIVENESS OF STEM CELL THERAPY IN MYOCARDIAL INFARCTION BASED ON ENZYME ACTIVITY AND LIVER HISTOPATOLOGY

This research aims to study the effectiveness of single placenta stem cell therapy (ST) or a combination of cardiomyocyte co-cultures in pigs with myocardial infarction based on enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity as well as liver histopathology. Nine pigs were divided into three treatment groups (K1, K2, and K3) consisting of three pigs per group. Blood sampling was performed one-hour pre-surgery, post-ST-elevation, and eight weeks post-therapy. The experimental animals were treated for eight weeks, then euthanized and necropsied for liver removal. ALT and AST values were analyzed using one-way analysis of variance (ANOVA) and followed by Tukey's test. Histopathological changes in liver tissue were analyzed using Image J 1.53 software, and the measured data were analyzed using oneway ANOVA. The results showed an increase in the ALT and AST values of the three treatment groups on post-ST-elevation blood sampling but were still within normal limits except for K1. ALT and AST values decreased significantly (P0.05) in the group treated with single placental stem cell (K2) or combined cardiomyocyte co-culture (K3). The ALT value of K3 decreased but not significantly (P0.05) compared to K2. The AST value of K3 decreased significantly (P0.05) but was not significantly different (P0.05) compared to K2. The liver tissue showed circulatory disturbances in the central venous congestion, portal vein, sinusoidal dilatation and congestion, and hepatocyte changes in the form of centrilobular necrosis and hydropic degeneration. Stem cell therapy alone (K2) or in combination with cardiomyocyte co-culture (K3) had similar percentage of centrilobular necrosis which was significantly lower than K1 (P0.05). Hepatocyte hydropic degeneration on K2 was classified as mild, whereas on K3 was classified as moderate. As conclusion, administration of placental stem cell therapy alone or in combination with cardiomyocyte co-culture can reduce ALT and AST values and could repair liver tissue.

Inhibition of soluble epoxide hydrolase reverses bone loss in periodontitis by upregulating EMCN and inhibiting osteoclasts

BackgroundImproving the microenvironment to augment endogenous regenerative potential has emerged as a fundamental concept for stimulating and expediting periodontal tissue repair and regeneration. Previous studies have demonstrated that TPPU, a soluble epoxide hydrolase inhibitor (sEHi), mediates the suppression of inflammatory bone loss in periodontitis models. However, the underlying mechanisms remain largely elusive.MethodsIn this study, we constructed a human umbilical vein endothelial cell (HUVEC) and periodontal ligament stem cell (PDLSC) coculture system in vitro and tested the anti-inflammatory effect of TPPU under inflammatory conditions. The roles of HIF-1α and Endomucin (EMCN) in the anti-inflammatory effects of TPPU were analyzed. The effects of TPPU on osteogenesis and osteoclastogenesis in cocultured cells were examined. The in vivo periodontitis model further verified the effects of TPPU on inhibiting neutrophil adhesion and inflammation and inhibiting osteoclasts.ResultsOur in vitro experiments demonstrated that TPPU enhances the interaction between mesenchymal stem cells and vascular endothelial cells to enhance anti-inflammatory and osteogenic differentiation effects and revealed a new anti-inflammatory mechanism of TPPU involving the upregulation of EMCN in endothelial cells to prevent lymphocyte recruitment. We also confirmed that TPPU inhibits osteoclast activity. Our in vivo findings showed that TPPU inhibits osteoclast activity and neutrophil adhesion and enhances periodontal tissue repair and regeneration.ConclusionsTPPU promotes local regeneration in periodontitis by inhibiting inflammation and bone resorption. Thus, targeting soluble epoxide hydrolase represents an endogenous regenerative strategy for periodontitis treatment.

Engineering Biomimetic Microvascular Capillary Networks in Hydrogel Fibrous Scaffolds via Microfluidics-Assisted Co-Axial Wet-Spinning.

The microvascular bed plays a crucial role in establishing nutrient exchange and waste removal, as well as maintaining tissue metabolic activity in the human body. However, achieving microvascularization of engineered 3D tissue constructs is still an unsolved challenge. In this work, we developed biomimetic cell-laden hydrogel microfibers recapitulating oriented microvascular capillary-like networks by using a 3D bioprinting technique combined with microfluidics-assisted coaxial wet-spinning. Highly packed and aligned bundles embedding a coculture of human bone marrow-derived mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were produced by simultaneously extruding two different bioinks. To this aim, core-shell fibers were wet-spun in a coagulation bath to collect the scaffolds later on a rotary drum. Initially, the versatility of the proposed system was assessed for the extrusion of multimaterial core-shell hydrogel fibers. Subsequently, the platform was validated for the in vitro biofabrication of samples promoting optimal cell alignment along the fiber axis. After 3 weeks of culture, such fiber configuration resulted in the development of an oriented capillary-like network within the fibrin-based core and in the endothelial-specific CD31 marker expression upon MSC/HUVEC maturation. Synergistically, the vertical arrangement of the coaxial nozzle coupled with the rotation of the fiber collector facilitated the rapid creation of tightly packed bundles characterized by a dense, oriented, and extensively branched capillary network. Notably, such findings suggest that the proposed biofabrication strategy can be used for the microvascularization of tissue-specific 3D constructs.

Immunocompetent Brain Organoids with Microglia Allow Advanced Aging Research.

Aging is a complex and multifactorial process that significantly affects brain function and health, since it is commonly associated with the emergence of neurodegenerative diseases. Recent advances in stem cell technology have facilitated the development of brain organoids, three-dimensional structures that mimic key aspects of brain architecture and functionality. By incorporating microglia, the resident monocyte-derived immune cells of the central nervous system, immunocompetent brain organoids can provide a more physiologically relevant model for studying brain aging. This chapter explores the methodology of immunocompetent brain organoids for advanced aging research, detailing protocols for their generation from a co-culture of neural stem cells and primitive macrophage progenitors.

The regenerative capacity of hepatocyte organoids following long-term cryopreservation in Republic of Korea

Background: Hepatocyte organoids (HOs) are more functionally versatile than bile duct epithelial cell organoids, but have a shorter lifespan. This limitation has been improved by optimizing culture methods, but could remain a barrier to their wider application in research and therapy, especially for use at the appropriate time.Methods: This study aimed to prolong the lifespan of pig HOs and assess their viability and functionality after a year of cryopreservation. Genes involved in apoptosis (TP53, P21, CASP8) and liver function (ALB, CYP3A29, EPCAM) were analyzed to evaluate the impact of adipose-derived mesenchymal stem cell (A-MSC) co-culture before and after freezing on cryoresistance. HOs were cut into fragments, cryopreserved for a year, and cultured alone or co-cultured with A-MSCs in Matrigel post-thawing.Results: After thawing, co-cultured HOs exhibited a higher development rate than those cultured alone. P21 expression increased irrespective of pre-freezing culture conditions. The ALB and CYP3A29 expression patterns resembled those of non-frozen HOs, with similar effects from co-culture. EPCAM expression surged post-freezing.Conclusion: This study demonstrates that HOs maintain liver function post-preservation, showing increased EPCAM expression and enhanced regenerative capacity against cryoinjury. These findings suggest that HOs may be a valuable cell source for drug development in animals and research on medications for human diseases.

Mesenchymal Stem Cell and Hematopoietic Stem and Progenitor Cell Co-Culture in a Bone-Marrow-on-a-Chip Device toward the Generation and Maintenance of the Hematopoietic Niche.

Bone marrow has raised a great deal of scientific interest, since it is responsible for the vital process of hematopoiesis and is affiliated with many normal and pathological conditions of the human body. In recent years, organs-on-chips (OoCs) have emerged as the epitome of biomimetic systems, combining the advantages of microfluidic technology with cellular biology to surpass conventional 2D/3D cell culture techniques and animal testing. Bone-marrow-on-a-chip (BMoC) devices are usually focused only on the maintenance of the hematopoietic niche; otherwise, they incorporate at least three types of cells for on-chip generation. We, thereby, introduce a BMoC device that aspires to the purely in vitro generation and maintenance of the hematopoietic niche, using solely mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs), and relying on the spontaneous formation of the niche without the inclusion of gels or scaffolds. The fabrication process of this poly(dimethylsiloxane) (PDMS)-based device, based on replica molding, is presented, and two membranes, a perforated, in-house-fabricated PDMS membrane and a commercial poly(ethylene terephthalate) (PET) one, were tested and their performances were compared. The device was submerged in a culture dish filled with medium for passive perfusion via diffusion in order to prevent on-chip bubble accumulation. The passively perfused BMoC device, having incorporated a commercial poly(ethylene terephthalate) (PET) membrane, allows for a sustainable MSC and HSPC co-culture and proliferation for three days, a promising indication for the future creation of a hematopoietic bone marrow organoid.

Establishment of an in vitro choroid complex system for vascular response screening

The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases.

Formation of ovarian organoid by co-culture of human endometrial mesenchymal stem cells and mouse oocyte in 3-dimensional culture system.

The online version contains supplementary material available at 10.1007/s10616-024-00639-w.

Cell Instructive Behavior of Composite Scaffolds in a Co-Culture of Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Assembled/Disassembled Modular Scaffolds for Multicellular Tissue Engineering.

The behavior of tissue resident cells can be influenced by the spatial arrangement of cellular interactions. Therefore, it is of significance to precisely control the spatial organization of various cells within multicellular constructs. It remains challenging to construct a versatile multicellular scaffold with ordered spatial organization of multiple cell types. Herein, a modular multicellular tissue engineering scaffold with ordered spatial distribution of different cell types is constructed by assembling varying cell-laden modules. Interestingly, the modular scaffolds can be disassembled into individual modules to evaluate the specific contribution of each cell type in the system. Through assembling cell-laden modules, the macrophage-mesenchymal stem cell (MSC), endothelial cell-MSC, and chondrocyte-MSC co-culture models are successfully established. The in vitro results indicate that the intercellular cross-talk can promote the proliferation and differentiation of each cell type in the system. Moreover, MSCs in the modular scaffolds may regulate the behavior of chondrocytes through the nuclear factor of activated T-Cells (NFAT) signaling pathway. Furthermore, the modular scaffolds loaded with co-cultured chondrocyte-MSC exhibit enhanced regeneration ability of osteochondral tissue, compared with other groups. Overall, this work offers a promising strategy to construct a multicellular tissue engineering scaffold for the systematic investigation of intercellular cross-talk and complex tissue engineering.

Effects of Semiconductor Laser Irradiation on Differentiation of Human Dental Pulp Stem Cells in Co-Culture with Dentin.

This study aimed to determine the effect of photobiomodulation therapy induced by semiconductor laser irradiation on human dental pulp stem cell (hDPSC) proliferation and their differentiation into odontoblast-like cells (OLCs). The effects of various semiconductor laser irradiation conditions on hDPSCs were examined. Three groups were evaluated: a single laser irradiation at 6 h post-seeding, multiple laser irradiations up to four times every 4 days after the first dose, and a control with no laser irradiation. The cells were irradiated at 10, 30, and 150 mW using a semiconductor laser. The effect of laser irradiation on hDPSC differentiation into OLCs was also determined. Four groups were evaluated, including co-culture using basic medium and dentin discs, simple culture using OLC differentiation-inducing medium, co-culture using OLC differentiation-inducing medium and dentin discs, and control culture with basic medium. The expression of the nestin, ALP, DSPP, and DMP-1 genes was measured using real-time PCR. The multiple irradiation group irradiated at 30 mW exhibited significantly more cell proliferation than the control. The expression of nestin associated with differentiation into OLCs during each culture period tended to be lower, whereas DSPP and ALP expression was higher compared with that of the control. Multiple laser irradiations at a low power of 30 mW induced significant hDPSC proliferation and might induce differentiation into OLCs.

Periostin in Cancer-Associated Fibroblasts Promotes Esophageal Squamous Cell Carcinoma Progression by Enhancing Cancer and Stromal Cell Migration

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment are involved in the progression of various cancers, including esophageal squamous cell carcinoma (ESCC). CAF-like cells were generated through direct co-culture of human bone marrow-derived mesenchymal stem cells, one of CAF origins, with ESCC cells. Periostin (POSTN) was found to be highly expressed in CAF-like cells. After direct co-culture, ESCC cells showed increased malignant phenotypes, such as survival, growth, and migration, as well as increased phosphorylation of Akt and extracellular signal-regulated kinase (Erk). Recombinant human POSTN activated Akt and Erk signaling pathways in ESCC cells, enhancing survival and migration. The suppression of POSTN in CAF-like cells by siRNA during direct co-culture also suppressed enhanced survival and migration in ESCC cells. In ESCC cells, knockdown of POSTN receptor integrin β4 inhibited Akt and Erk phosphorylation, and survival and migration increased by POSTN. POSTN also enhanced mesenchymal stem cell and macrophage migration and endowed macrophages with tumor-associated macrophage-like properties. Immunohistochemistry showed that high POSTN expression in the cancer stroma was significantly associated with tumor invasion depth, lymphatic and blood vessel invasion, higher pathologic stage, CAF marker expression, and infiltrating tumor-associated macrophage numbers. Moreover, patients with ESCC with high POSTN expression exhibited poor postoperative outcomes. Thus, CAF-secreted POSTN contributed to tumor microenvironment development. These results indicate that POSTN may be a novel therapeutic target for ESCC.

Influential factors for optimizing and strengthening mesenchymal stem cells and hematopoietic stem cells co-culture.

Mesenchymal stem cells (MSCs) and Hematopoietic stem cells (HSCs) are two types of bone marrow stem cells that can proliferate and differentiate into different cell lineages. HSCs interact with MSCs under protective conditions, called niche. Numerous studies have indicated supportive effects of MSCs on HSCs proliferation and differentiation. Furthermore, HSCs have many clinical applications and could treat different hematologic and non-hematologic diseases. For this purpose, there is a need to perform in vitro studies to optimize their expansion. Therefore, various methods including co-culture with MSCs are used to address the limitations of HSCs culture. Some parameters that might be effective for improving the MSC/ HSC co-culture systems. Manipulating culture condition to enhance MSC paracrine activity, scaffolds, hypoxia, culture medium additives, and the use of various MSC sources, have been examined in different studies. In this article, we investigated the potential factors for optimizing HSCs/ MSCs co-culture. It might be helpful to apply a suitable approach for providing high-quality HSCs and improving their therapeutic applications.

ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells.

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Implementation of the neuro-glia-vascular unit through co-culture of adult neural stem cells and vascular cells and transcriptomic analysis of diverse Aβ assembly types

BackgroundThe blood-brain barrier (BBB) is a specialized layer between blood vessels and tissue in the brain, which is comprised of a neuro-glia-vascular (NGV) unit, thus play a vital role in various brain diseases. New methodWe developed the in vitro NGV units by co-culturing brain microvascular endothelial cells (BMECs; bEnd.3) and primary neural stem cells extracted from subventricular zone of adult mice. This approach was designed to mimic the RNA profile conditions found in the microvessels of a mouse brain and confirmed through various comparative transcriptome analyses. ResultsOptimal NGV unit development was achieved by adjusting cell density-dependent co-culture ratios. Specifically, the morphogenic development and neuronal association of astrocyte endfeet were well observed in the contact region with BMECs in the NGV unit. Through transcriptome analysis, we compared co-cultured bEnd.3/NSCs with monocultured bEnd.3 or NSCs and additionally compared them with previously reported mouse brain vascular tissue to show that this NGV unit model is a suitable in vitro model for neurological disease such as Alzheimer’s disease (AD). Comparison with existing method(s)This in vitro NGV unit was formed from neural stem cells and vascular cells in the brain of adult mice, not embryos. It is very useful for studying brain disease mechanisms by identifying proteins and genes associated with diseases progress. ConclusionsWe suggest that this simple in vitro NGV model is appropriate to investigate the relationship between BBB changes and pathological factors in the fields of neurovascular biology and cerebrovascular diseases including AD.

Sustained Release of Dexamethasone from 3D-Printed Scaffolds Modulates Macrophage Activation and Enhances Osteogenic Differentiation.

Enhancing osteogenesis via modulating immune cells is emerging as a new approach to address the current challenges in repairing bone defects and fractures. However, much remains unknown about the crosstalk between immune cells and osteolineage cells during bone formation. Moreover, biomaterial scaffold-based approaches to effectively modulate this crosstalk to favor bone healing are also lacking. This study is the first to investigate the interactions between macrophages and mesenchymal stem cells (MSCs) in co-cultures with the sustained release of an anti-inflammatory and pro-osteogenesis drug (dexamethasone) from three-dimensional (3D)-printed scaffolds. We successfully achieved the sustained release of dexamethasone from polycaprolactone (PCL) by adding the excipient-sucrose acetate isobutyrate (SAIB). Dexamethasone was released over 35 days in the 17-163 nM range. The osteogenic differentiation of MSCs was enhanced by M1 macrophages at early time points. The late-stage mineralization was dominated by dexamethasone, with little contribution from the macrophages. Besides confirming BMP-2 whose secretion was promoted by both dexamethasone and M1 macrophages as a soluble mediator for enhanced osteogenesis, IL-6 was found to be a possible new soluble factor that mediated osteogenesis in macrophage-MSC co-cultures. The phenotype switching from M1 to M2 was drastically enhanced by the scaffold-released dexamethasone but only marginally by the co-cultured MSCs. Our results offer new insight into macrophage-MSC crosstalk and demonstrate the potential of using drug-release scaffolds to both modulate inflammation and enhance bone regeneration.

Investigation of the optimal light parameters for photobiomodulation to induce osteogenic differentiation of the human bone marrow stem cell and human umbilical vein endothelial cell co-culture.

Bones have an important role in the human body with their complex nature. Mesenchymal stem cells and endothelial cells together support their unique and complex nature. Photobiomodulation (PBM) is a promising method that provides cell proliferation, osteogenic differentiation, and bone regeneration. However, there are still unknowns in the mechanism of osteogenic differentiation induced by PBM. The main aim of the study is to understand the molecular mechanism of PBM at 655 and 808nm of wavelengths and identify the most effective energy densities of both wavelengths for osteogenic differentiation. The effect of PBM on osteogenic differentiation of Human Bone Marrow Stem Cell (hBMSC) and Human Umbilical Vein Endothelial Cell (HUVEC) co-culture was examined at 1, 3, and 5J/cm2 energy densities of red and near-infrared light through different analysis such as cell viability, scratch assay, intracellular reactive oxygen species production, and ATP synthesis, nitric oxide release, temperature monitoring, and osteogenic differentiation analyses. Even though all PBM-treated groups exhibited better results compared to the control group, 5J/cm2 energy density induced faster cell proliferation and migration at both wavelengths. The increases in ATP and NO levels as signaling molecules, and the increases in DNA, ALPase, and calcium contents as osteogenic markers were higher in the groups treated with 5J/cm2 energy density at both wavelengths. Only a slight change was obtained in the level of intracellular ROS after any light applications. It can be concluded that NO release has a very important role together with ATP production in PBM therapy to trigger DNA synthesis, ALPase activity, and mineralization for osteogenic differentiation of the hBMSC and HUVEC co-culture at 655 and 808nm of wavelengths.

Preparation and in vitro osteogenic evaluation of high-strength ceramic artificial bone loaded with anti-tuberculosis drug PaMZ

The objective of this study was to prepare a high-strength ceramic artificial bone loaded with the anti-tuberculosis drug PaMZ (delamanid, moxifloxacin, and pyrazinamide) and evaluate its physical characteristics and osteogenic potential. We utilized 3D printing technology to fabricate artificial bones and then obtained a high-strength ceramic artificial bone by high-temperature firing. Then, a triple combination of anti-tuberculosis drugs, including delamanid (Pa), moxifloxacin (M), pyrazinamide (Z), and polylactic acid-co-glycolic acid mixed in a ratio of 3:12:45:140, was incorporated onto the surface of the ceramic artificial bone. Consequently, a high-strength ceramic artificial bone, loaded with anti-tuberculosis drugs, was successfully obtained. The physical characteristics of the drug-loaded artificial bone were assessed using an electronic universal testing machine and scanning electron microscopy. The osteogenic performance of the artificial bone was evaluated through rat bone marrow mesenchymal stem cell (rBMSCs) co-culture experiment, cell counting kit-8 (CCK-8) cell proliferation assay, alkaline phosphatase staining, and alizarin red staining. The drug-loaded ceramic artificial bone exhibited favorable physical characteristics, void interconnection, a porosity of 30.6% ± 0.7%, and a compressive strength of 17.65 ± 0.46 MPa. The rBMSCs co-culture experiment and CCK-8 cell proliferation experiment demonstrated excellent cell compatibility, while alkaline phosphatase and alizarin red staining indicated good in vitro osteogenic performance. In summary, the high-strength ceramic artificial bone loaded with the anti-tuberculosis drug PaMZ exhibited a favorable morphological structure and compressive strength. In addition, it demonstrated good biocompatibility and osteogenic properties.